In Cellulo Bioorthogonal Catalysis by Encapsulated AuPd Nanoalloys: Overcoming Intracellular Deactivation

Bioorthogonal metallocatalysis has opened up a xenobiotic route to perform nonenzymatic catalytic transformations in living settings. Despite their promising features, most metals are deactivated inside cells by a myriad of reactive biomolecules, including biogenic thiols, thereby limiting the catalytic functioning of these abiotic reagents. Here we report the development of cytocompatible alloyed AuPd nanoparticles with the capacity to elicit bioorthogonal depropargylations with high efficiency in biological media. We also show that the intracellular catalytic performance of these nanoalloys is significantly enhanced by protecting them following two different encapsulation methods. Encapsulation in mesoporous silica nanorods resulted in augmented catalyst reactivity, whereas the use of a biodegradable PLGA matrix increased nanoalloy delivery across the cell membrane. The functional potential of encapsulated AuPd was demonstrated by releasing the potent chemotherapy drug paclitaxel inside cancer cells. Nanoalloy encapsulation provides a novel methodology to develop nanoreactors capable of mediating new-to-life reactions in cells.

I n the early 2010s, the paths of bioorthogonal chemistry and nanotechnology crossed at the very center of the periodic table of elements, 1,2 sparking the emergence of transition-metal catalysts (TMCs) as bioorthogonal nanotools. 3,4 Over a decade later, numerous biocompatible TMC-based strategies with different features and functions have been developed for various biomedical applications, including de novo enzyme design, 5,6 labeling/uncaging of biomolecules, 7,8 or the release of metal-activated probes and therapeutics. 9,10 Organometallic complexes, 11−15 artificial metalloproteins, 5,6,16,17 nanozymes and MOFs, 18−27 metal-loaded exosomes and macrophages, 28 The size and nature of the ligands or scaffolds bound to the metal atoms or NPs will dictate whether these nanoreactors are to exert their function in the extracellular or intracellular space, with each environment facing different conditions. While the interstitial liquid is mostly composed of water (up to 95%), intracellular bioorthogonal catalysis is severely restricted by biomolecule crowding (>20% protein by weight 37 ) and the reductive environment of the cell cytoplasm. Under such stringent conditions, most TMCs are poisoned in relatively short periods of time. For this reason, technologies that protect abiotic metals from thiol-rich biomolecules and other reactive species and, at the same time, facilitate access of the substrate to metal active sites are in demand to overcome this issue.
A number of research laboratories have investigated the use of different TMCs (including noble metals such as Ru, Pd, Au, and Pt) and reactions (e.g., dealkylations, cross-couplings, cycloadditions, and ring-closure metathesis) in cells and organisms with the aim of expanding the chemical toolbox for bioorthogonal metallocatalysis. Depropargylations are one such reactions. O-and N-propargyl groups have been extensively used as a masking strategy in chemical biology and medicinal chemistry to render bioactive agents inactive, while activatable by abiotic metal catalysis. 10,21,28,32−35,38−44 Because of the lack of natural "depropargylases", this strategy offers superb control over bioorthogonal dissociative processes, enabling selective uncaging of drugs exclusively in the presence of a metal activator, even in vivo. 21,34,44 Pd and Au NPs stand out among the metallic nanocatalysts that catalyze depro-pargylation reactions. Interestingly, there has been an oversight of the reactivity of alloys of these or other noble metals so far. Encouraged by the potential benefits offered by alloyed nanomaterials, 45 we embarked on a systematic study to investigate the capacity of a range of single-metal NPs and alloys to uncage a propargyl-masked prodye (PocRho) 10 under physiological conditions.
Single-metal NPs of Pd, Au, Pt, and Ru and their corresponding bimetallic alloys were manufactured in a single step using tetrakis(hydroxymethyl)-phosphonium chloride as a simultaneous reducing agent and stabilizing ligand. 46−48 (See the full experimental details in the Supporting Information.) NPs were then incubated with PocRho, a bis-propargyloxycarbonyl-protected prodye that releases green fluorescent rhodamine 110 (Rho) upon double depropargylation. 21 Reactions were run at 37°C in PBS in the absence and presence of serum to determine which metallic NPs were compatible not only with physiological media but also with supplements required in cell culture. An analysis of fluorescence intensity was carried out using a spectrofluorometer (PerkinElmer EnVision, λ ex/em 480/535 nm). As shown in Figure 1a, single-metal Pd NPs and Pd-containing nanoalloys exhibited superior capabilities to uncage Rho, highlighting the performance of AuPd in serum-containing media. (See the kinetics study for best-performing NPs in Figure S1, Supporting Information.) Next, we tested the tolerability of human lung adenocarcinoma A549 cells to treatment with the most catalytic NPs identified in the fluorogenic study. Notably, AuPd nanoalloys induced no change in cell viability at any of the concentrations tested, whereas the rest of the NPs displayed variable levels of toxicity at 20 μg/mL (Figure 1b).
Encouraged by the catalytic properties and tolerability of AuPd, we performed a preliminary in cellulo study to test the bioorthogonal reactivity of the NPs in A549 cells. Disappointingly, cancer cells pretreated with AuPd were not able to convert an inactive precursor of paclitaxel into its toxic form, indicating that these NPs are either unable to enter cells or rapidly deactivated in the cell cytoplasm. The latter is not surprising as metallic Au has a high affinity for thiol groups, 32 which are ubiquitous in proteins and can bind to Au surfaces, sterically hindering the access of the substrate to the catalytic sites. Consequently, we decided to investigate different nanoencapsulation methods with the aim of enhancing nanoalloy delivery and protecting the metal NPs from direct contact with thiol-rich biomolecules in the crowded cell cytoplasm. Two kinds of materials were tested to encapsulate AuPd: poly(lactic-co-glycolic acid) (PLGA) and mesoporous silica nanorods (SiO 2 ). Direct encapsulation of preformed AuPd NPs in the PLGA emulsion results in an inconsistent nanoalloy load distribution, 49 making this method inadequate for our goals. Therefore, AuPd-PLGA was prepared by the water/oil/water emulsion and solvent evaporation method using a new methodology inspired by a recently reported procedure designed to produce Pd nanosheets in situ in PLGA. 50 (See Figure S2 and experimental details in the Supporting Information.) Images from transmission electron microscopy (TEM and HAADF-STEM) clearly show 2 to 3 nm AuPd NPs embedded inside PLGA nanodrops of approximately 100 nm in diameter (Figure 1c,d and Figure  S3). As shown in Figure 1c, the AuPd NPs were selectively loaded in each nanomatrix of PLGA, which is evidence of the efficiency of the loading method. Elemental analysis confirmed the alloyed composition of the AuPd NPs (Figure 1d), which demonstrates the versatility of the in situ approach to yield not only single-metal NPs 48 but also AuPd nanoalloys, being the first procedure that facilitates the crystallization of alloyed NPs in double emulsions of PLGA. On the other hand, ordered mesoporous SiO 2 nanorods were prepared according to previously reported procedures, 52,53 followed by amine-grafting with 3-aminopropyl-triethoxysilane and loading of preformed The PocRho fluorogenic test was then used to compare the catalytic efficacy of uncoated AuPd NPs versus nanoencapsulated AuPd at 37°C in PBS with and without serum. Analysis revealed that AuPd SiO 2 mediated the highest fluorescence signal, being superior to uncoated AuPd nanoalloys ( Figure 2). This may be associated with the homogeneous dispersion of AuPd NPs throughout the pore channels of the scaffold. 51 In contrast, AuPd PLGA demonstrated a significantly lower depropargylation capacity at equal metal concentrations. Subsequently, we studied the

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pubs.acs.org/NanoLett Letter capacity of each of the materials to deliver AuPd into A549 cells. Two methods were used to study nanoalloy transcellular delivery: TEM, to visualize the metal presence in the cell cytoplasm, and inductively coupled plasma-atomic emission spectrometry (ICP-OES), for quantitative metal content analysis. Cells were treated with nanoencapsulated AuPd NPs for 30 min. After the media containing NPs were removed and the adhered cells were washed twice with PBS, cells were detached by trypsinization and centrifuged to further eliminate extracellular NPs by discarding the supernatant. Cells were then incubated on a coverslip for an additional 24 h, fixed (paraformaldehyde), and processed for TEM analysis. Rather than being adsorbed on the cell membrane, images verified the presence of AuPd PLGA and AuPd SiO 2 in the cytoplasm of A549 cells, which appeared as separate NPs of various sizes across the cell cytoplasm (Figure 3a,b; see additional images in Figure S7, Supporting Information). On the other hand, an intracellular metal content study was carried out by incubating A549 cells with freestanding AuPd NPs, AuPd PLGA, and AuPd SiO 2 (20 μg/mL of metal content), followed by the same procedure described above to remove extracellular NPs and thus ensure that only internalized nanoalloys were measured. Cell pellets were digested (10% HNO 3 ) and analyzed by ICP-OES. As shown in Figure 3c, the content of Au and Pd in cells treated with uncoated AuPd was relatively low, which may in part explain the lack of capacity of these NPs to activate prodrugs inside cells. Notably, encapsulated AuPd achieved far superior intracellular delivery, highlighting the performance of AuPd PLGA, whose capacity to deliver the nanoalloys into A549 cells was approximately 25% higher than that of AuPd SiO 2 . As expected, the Au/Pd content ratio remained constant for the three nanocomposites. Encouraged by the improved catalytic properties of AuPd SiO 2 in biological media and the superior transcellular carrier abilities of AuPd PLGA, an intracellular prodrug activation study was performed using the inactive chemotherapy precursor Pro-PTX (Figure 4a), which upon a single depropargylation reaction triggers a self-immolation cascade that releases highly toxic PTX (mechanism described in a previous work 35 ). A549 cells were preincubated with either freestanding or encapsulated AuPd NPs (20 μg of metal/mL) for 30 min and then washed twice with PBS to eliminate extracellular nanoalloys. Vehicle (0.1% v/v DMSO) or Pro-PTX (1 μM) was added to the cells and incubated for 5 days. Cell viability was measured using the PrestoBlue assay. PTX (1 μM) treatment and untreated cells (0.1% v/v DMSO) were used as positive and negative controls, respectively. Cells treated with Pro-PTX (1 μM) in the absence of AuPd NPs were used as an additional negative control. As shown in Figure 4b, A549 cell proliferation was unaffected by treatment with either Pro-PTX or the AuPd nanoalloys. In contrast, the incubation of Pro-PTX with cells pretreated with encapsulated AuPd elicited highly potent inhibition of cancer cell proliferation, showing anticancer activity comparable to the direct treatment with PTX. Remarkably, the intracellular drug uncaging capacities of both encapsulation methods were essentially equivalent. This indicates that the superior delivery properties of AuPd PLGA compensates for its lower catalytic properties relative to those of AuPd SiO 2 . Of note, incubation of Pro-PTX with cells pretreated with "naked" AuPd NPs did not show any reduction of cell viability, in agreement with their reduced cellular internalization (as shown in Figure 3c). This study demonstrates that the encapsulation of metal nanoalloys can serve to improve both catalytic and cell delivery properties, thereby enabling the performance of intracellular bioorthogonal reactions.
Finally, to validate that the combined treatment of encapsulated AuPd NPs and Pro-PTX results in the same

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Letter antiproliferative mode of action as for the parent drug PTX, we studied microtubule stabilization by immunofluorescence. 54 Cells were treated as previously described; fixed after 2 days of treatment; incubated with DAPI (cell nuclei stain), anti-αtubulin IgG (for microtubules), and TRITC-phalloidin (for actin filaments); and imaged by confocal microscopy (Olympus FV1000). As shown in Figure 4c, negative controls did not induce changes in cell morphology. In contrast, treating A549 cells with PTX led to microtubule accumulation (green channel), round-shaped cells, and fragmented nuclei (independent channel images are shown in Figure S8, Supporting Information). Notably, equivalent morphological changes were observed in cells treated with both Pro-PTX and encapsulated AuPd NPs, evidence that the anticancer effect mediated by these combinations is the result of the intracellular generation of PTX.
In conclusion, we have studied the capacity of noble metal NPs (Pd, Au, Pt, and Ru, and their corresponding bimetallic alloys) to mediate depropargylation reactions in biological media and discovered that AuPd nanoalloys display superior catalytic properties and tolerability compared to any other single-metal or alloyed NP tested in this work. The enhanced catalysis elicited by alloyed AuPd NPs is likely a consequence of a combination of factors, including geometric and electronic effects on the NP surface 55 and the fact that each metal mediates depropargylation reactions by different but complementary mechanisms. 32,38 Regrettably, these bimetallic NPs show negligible bioorthogonal reactivity inside cells. To improve their in cellulo performance, we investigated two nanoencapsulation methods, both of which successfully enabled AuPd-mediated intracellular uncaging of the clinically approved drug PTX. Notably, each material promoted AuPd catalysis by different means: nondegradable mesoporous silica nanorods augmented the catalytic performance of the nanoalloys, whereas biodegradable PLGA matrixes enhanced transcellular NP delivery. By protecting AuPd NPs with scaffolds with distinct features, this investigation provides a novel and versatile strategy for protecting metal NPs and performing intracellular biorthogonal catalysis toward different applications, including the selective uncaging of probes and drugs inside cells.
Preparation and characterization of NPs, prodye, and prodrug; methods and experimental procedures; biological assays; and Figures

Notes
The authors declare no competing financial interest.